Synergistic activation of UCP-3 expression in cultured fetal rat brown adipocytes by PPARalpha and PPARgamma ligands

Rat brown adipocytes express mRNAs for Uncoupling Proteins (UCP) 1, 2 and 3 and the Peroxisome Proliferator Activated Receptors (PPAR) alpha and gamma. We have examined the effects of selective PPARalpha or -gamma activation on changes in UCP-1 and UCP-3 mRNA levels in cultured fetal rat brown adipocytes (FBA). Rosiglitazone (1.0 microM), a selective PPARgamma agonist, elicited 5- and 3-fold increases in UCP-1 and UCP-3, respectively. The PPARalpha ligand, Wy14643 (10.0 microM) increased UCP-3 tenfold, but decreased UCP-1. A synergistic effect on UCP-3 expression (30-fold increase; P < 0. 05) was observed when FBA were exposed to a combination of Wy14643 (10.0 microM) and rosiglitazone (10.0 microM). Thus, activation of PPARgamma increases UCP-1 and UCP-3 levels which are differentially regulated by PPARalpha. A synergistic interaction occurs between PPARalpha and PPARgamma in the regulation of UCP-3 in FBA, probably via co-activator recruitment, suppression of co-repressor proteins or through a direct interaction at the level of the PPRE.     

4,4-Dimethyl-1,2,3,4-tetrahydroquinoline-based PPARalpha/gamma agonists. Part I: synthesis and pharmacological evaluation

Type-2 diabetes (T2D) is a complex metabolic disease characterized by insulin resistance in the liver and peripheral tissues accompanied by a defect in pancreatic beta-cell. Since their discovery three subtypes of Peroxisomes Proliferators Activated Receptors were identified namely PPARalpha, PPARgamma and PPARbeta/(delta). We were interested in designing novel PPARgamma selective agonists and/or dual PPARalpha/gamma agonists. Based on the typical topology of synthetic PPAR agonists, we focused our design approach on 4,4-dimethyl-1,2,3,4-tetrahydroquinoline as novel cyclic tail.     

A rapid, homogeneous, fluorescence polarization binding assay for peroxisome proliferator-activated receptors alpha and gamma using a fluorescein-tagged dual PPARalpha/gamma activator

Peroxisome proliferator-activated receptors (PPARs) and other members of the nuclear hormone receptor family are important drug targets for the treatment of metabolic diseases. PPARalpha and PPARgamma play crucial roles in lipid and glucose metabolism, respectively. Therefore, screening methods that help to rapidly identify activators of these receptors should be of considerable value. A homogeneous fluorescence polarization (FP) ligand binding assay capable of rapidly identifying ligands that bind to both PPARalpha and PPARgamma has been developed using purified PPARalpha or PPARgamma ligand binding domains and a fluorescein-labeled analog (FLA) of a potent dual PPARalpha/gamma activator. FLA activator showed good binding affinity toward both PPARalpha (K(i)=0.7microM) and PPARgamma (K(i)=0.4microM). The binding of FLA activator was rapid and reached a plateau within 10 min. The resulting FP signal was stable for at least 18h. The FP binding assay performed robustly in a 384-well format, and the average Z' value was 0.77. There was a good correlation between the binding potency (IC(50) values) and rank order of binding potency for a panel of standard PPAR ligands obtained in FP binding assay and scintillation proximity assay or gel filtration binding assays using (3)H-labeled PPARalpha (r(2)=0.99) and PPARgamma (r(2)=0.99) ligands. There was also a good correlation of IC(50) values obtained by FP binding assay and scintillation proximity assay for the clinically used PPAR activators. Thus, the FP binding assay with a single fluorescein-labeled PPARalpha/gamma dual activator offers a homogeneous nonradioactive, sensitive, robust, and less expensive high-throughput assay for detecting compounds that bind to both PPARgamma and PPARalpha. Using this FP binding assay, we have identified a large number of PPARalpha/gamma dual activators. A similar assay platform may be easily adapted to other members of the nuclear hormone receptor family.     

Dual PPARalpha /gamma activation provides enhanced improvement of insulin sensitivity and glycemic control in ZDF rats

Improvement of insulin sensitivity and lipid and glucose metabolism by coactivation of both nuclear peroxisome proliferator-activated receptor (PPAR)gamma and PPARalpha potentially provides beneficial effects over existing PPARgamma and alpha preferential drugs, respectively, in treatment of type 2 diabetes. We examined the effects of the dual PPARalpha/gamma agonist ragaglitazar on hyperglycemia and whole body insulin sensitivity in early and late diabetes stages in Zucker diabetic fatty (ZDF) rats and compared them with treatment with the PPARgamma preferential agonist rosiglitazone. Despite normalization of hyperglycemia and Hb A(1c) and reduction of plasma triglycerides by both compounds in both prevention and early intervention studies, ragaglitazar treatment resulted in overall reduced circulating insulin and improved insulin sensitivity to a greater extent than after treatment with rosiglitazone. In late-intervention therapy, ragaglitazar reduced Hb A(1c) by 2.3% compared with 1.1% by rosiglitazone. Improvement of insulin sensitivity caused by the dual PPARalpha/gamma agonist ragaglitazar seemed to have beneficial impact over that of the PPARgamma-preferential activator rosiglitazone on glycemic control in frankly diabetic ZDF rats.     

Design, synthesis, and structure-activity relationship of carbamate-tethered aryl propanoic acids as novel PPARalpha/gamma dual agonists

We have developed a new class of PPARalpha/gamma dual agonists, which show excellent agonistic activity in PPARalpha/gamma transactivation assay. In particular, (R)-9d was identified as a potent PPARalpha/gamma dual agonist with EC(50)s of 0.377 microM in PPARalpha and 0.136 microM in PPARgamma, respectively. Interestingly, the structure-activity relationship revealed that the stereochemistry of the identified PPARalpha/gamma dual agonists significantly affects their agonistic activities in PPARalpha than in PPARgamma.     

Regulation of p85alpha phosphatidylinositol-3-kinase expression by peroxisome proliferator-activated receptors (PPARs) in human muscle cells

Regulation of p85a phosphatidylinositol-3-kinase (p85alphaPI-3K) expression by peroxisome proliferator-activated receptor (PPAR) activators was studied in human skeletal muscle cells. Activation of PPARgamma or PPARbeta did not modify the expression of p85alphaPI-3K. In contrast, activation of PPARalpha increased p85alphaPI-3K mRNA. This effect was potentiated by 9-cis-retinoic acid, an activator of RXR. Up-regulation of p85alphaPI-3K gene expression resulted in a rise in p85alphaPI-3K protein level and in an increase in insulin-induced PI3-kinase activity. According to the role of p85alphaPI-3K in insulin action, these results suggest that drugs with dual action on both PPARgamma and PPARalpha can be of interest for the treatment of insulin resistance.     

PPARalpha /gamma ragaglitazar eliminates fatty liver and enhances insulin action in fat-fed rats in the absence of hepatomegaly

Peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma agonists lower lipid accumulation in muscle and liver by different mechanisms. We investigated whether benefits could be achieved on insulin sensitivity and lipid metabolism by the dual PPARalpha/gamma agonist ragaglitazar in high fat-fed rats. Ragaglitazar completely eliminated high-fat feeding-induced liver triglyceride accumulation and visceral adiposity, like the PPARalpha agonist Wy-14643 but without causing hepatomegaly. In contrast, the PPARgamma agonist rosiglitazone only slightly lessened liver triglyceride without affecting visceral adiposity. Compared with rosiglitazone or Wy-14643, ragaglitazar showed a much greater effect (79%, P < 0.05) to enhance insulin's suppression of hepatic glucose output. Whereas all three PPAR agonists lowered plasma triglyceride levels and lessened muscle long-chain acyl-CoAs, ragaglitazar and rosiglitazone had greater insulin-sensitizing action in muscle than Wy-14643, associated with a threefold increase in plasma adiponectin levels. There was a significant correlation of lipid content and insulin action in liver and particularly muscle with adiponectin levels (P < 0.01). We conclude that the PPARalpha/gamma agonist ragaglitazar has a therapeutic potential for insulin-resistant states as a PPARgamma ligand, with possible involvement of adiponectin. Additionally, it can counteract fatty liver, hepatic insulin resistance, and visceral adiposity generally associated with PPARalpha activation, but without hepatomegaly.     

20-carboxy-arachidonic acid is a dual activator of peroxisome proliferator-activated receptors alpha and gamma

20-carboxy-arachidonic acid (20-COOH-AA) is a metabolite of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid produced from arachidonic acid by cytochrome P450 (CYP) omega-oxidases. Alcohol dehydrogenases convert 20-HETE to 20-COOH-AA, and we now find that a microsomal preparation containing recombinant human CYP4F3B converts arachidonic acid to 20-HETE and 20-COOH-AA. Studies with transfected COS-7 cell expression systems indicate that 20-COOH-AA activates peroxisome proliferators-activated receptor (PPAR) alpha and PPARgamma. 20-COOH-AA was twice as potent as either 20-HETE or ciglitazone in stimulating PPARgamma-mediated luciferase expression. While 20-COOH-AA also was more potent than 20-HETE in increasing PPARalpha-mediated luciferase expression, the increase was only half as much as that produced by Wy-14643. 20-COOH-AA did not increase PPARalpha or PPARgamma expression in the transfected cells. Radiolabeled 20-COOH-AA was detected intracellularly when the COS-7 cells were incubated with either [3H]20-COOH-AA or [3H]20-HETE, and binding studies indicated that [3H]20-COOH-AA bound to the isolated ligand binding domains of PPARalpha (Kd=0.87+/-0.12 microM) and PPARgamma (Kd=1.7+/-0.5 microM). These findings suggest that 20-COOH-AA, a relatively stable metabolite of 20-HETE, might function as an endogenous dual activator of PPARalpha and PPARgamma.     

Combination therapy with PPARgamma and PPARalpha agonists increases glucose-stimulated insulin secretion in db/db mice

Although peroxisome proliferator-activated receptor (PPAR)gamma agonists ameliorate insulin resistance, they sometimes cause body weight gain, and the effect of PPAR agonists on insulin secretion is unclear. We evaluated the effects of combination therapy with a PPARgamma agonist, pioglitazone, and a PPARalpha agonist, bezafibrate, and a dual agonist, KRP-297, for 4 wk in male C57BL/6J mice and db/db mice, and we investigated glucose-stimulated insulin secretion (GSIS) by in situ pancreatic perfusion. Body weight gain in db/db mice was less with KRP-297 treatment than with pioglitazone or pioglitazone + bezafibrate treatment. Plasma glucose, insulin, triglyceride, and nonesterified fatty acid levels were elevated in untreated db/db mice compared with untreated C57BL/6J mice, and these parameters were significantly ameliorated in the PPARgamma agonist-treated groups. Also, PPARgamma agonists ameliorated the diminished GSIS and insulin content, and they preserved insulin and GLUT2 staining in db/db mice. GSIS was further increased by PPARgamma and -alpha agonists. We conclude that combination therapy with PPARgamma and PPARalpha agonists may be more useful with respect to body weight and pancreatic GSIS in type 2 diabetes with obesity.     

Promotion of adiponectin multimerization by emodin: A novel AMPK activator with PPARγ-agonist activity

Adiponectin is an important insulin‐sensitizing adipokine with multiple beneficial effects on obesity‐associated medical complications. It is secreted from adipocytes into circulation as high, medium, and low molecular weight forms (HMW, MMW, and LMW). Each oligomeric form of adiponectin exerts non‐overlapping biological functions, with the HMW oligomer possessing the most potent insulin‐sensitizing activity. In this study, we reported that emodin, a natural product and active ingredient of various Chinese herbs, activates AMPK in both 3T3‐L1 adipocytes and 293T cells. Activation of AMPK by emodin promotes the assembly of HMW adiponectin and increases the ratio of HMW adiponectin to total adiponectin in 3T1‐L1 adipocytes. Emodin might activate AMPK by an indirect mechanism similar to berberine. We also found that emodin activates PPARγ and promotes differentiation and adiponectin expression during differentiation of 3T3‐L1 preadipocytes. Therefore, emodin is a novel AMPK activator with PPARγ‐agonist activity. Our results demonstrate that the effects of emodin on adiponectin expression and multimerization are the ultimate effects resulting from both AMPK activation and PPARγ activation. The dual‐activity makes emodin or the derivatives potential drug candidates for the treatment of type 2 diabetes and other obesity‐related metabolic diseases. J. Cell. Biochem. 113: 3547–3558, 2012. © 2012 Wiley Periodicals, Inc.     

Expression of peroxisome proliferator-activated receptors alpha and gamma in differentiating human colon carcinoma Caco-2 cells

The expression of peroxisome proliferator-activated receptors alpha (PPARalpha) and gamma (PPARgamma) was studied in the human adenocarcinoma Caco-2 cells induced to differentiate by long term culture (15 days). The differentiation of Caco-2 cells was attested by increases in the activities of sucrase-isomaltase and alkaline phosphatase (two brush border enzymes), fatty acyl-CoA oxidase (AOX) and catalase (two peroxisomal enzymes), by an elevation in the protein levels of villin (a brush border molecular marker), AOX, peroxisomal bifunctional enzyme (PBE), catalase and peroxisomal membrane protein of 70 kDa (PMP70). and by the appearance of peroxisomes. The expression of PPARalpha and PPARgamma was investigated by Western blotting, immunocytochemistry, Northern blotting and S1 nuclease protection assay during the differentiation of Caco-2 cells. The protein levels of PPARalpha, PPARgamma, and PPARgamma2 increased gradually during the time-course of Caco-2 cell differentiation. Immunocytochemistry revealed that PPARalpha and gamma were localized in cell nuclei. The PPARgamma1 protein was encoded by PPARgamma3 mRNA because no signal was obtained for PPARgamma1 mRNA using a specific probe in S1 nuclease protection assay. The amount of PPARgamma3 mRNA increased concomitantly to the resulting PPARgamma1 protein. On the other hand, the mRNA of PPARalpha and PPARgamma2 were not significantly changed, suggesting that the increase in their respective protein was due to an elevation of the translational rate. The role played by the PPAR subtypes in Caco-2 cell differentiation is discussed.     

Safety issues and prospects for future generations of PPAR modulators

Because of their wide range of actions on glucose homeostasis, lipid metabolism and vascular inflammation, peroxisome proliferator-activated receptors (PPARs) are promising targets for the development of new drugs for the treatment of metabolic disorders such as diabetes, dyslipidemia and atherosclerosis. In clinical practice, PPARalpha agonists, such as the already available fibrates, improve dyslipidemia, while PPARgamma agonists, such as thiazolidinediones, improve insulin resistance and diabetes. The complementary action of simultaneous activation of each PPAR in patients suffering from metabolic syndrome and type 2 diabetes has led to new pharmacological strategies focused on the development of agonists targeting more than one receptor such as the dual PPARalpha/gamma agonists. However, despite the proven benefits of targeting PPARs, safety concerns have recently led to late stage development failures of various PPAR agonists including novel specific PPARgamma agonists and dual PPARalpha/gamma agonists. These safety concerns include potential carcinogenicity in rodents, signs of myopathy and rhabdomyolysis, increase in plasma creatinine and homocysteine, weight gain, fluid retention, peripheral edema and potential increased risk of cardiac failure. Although the discontinued compounds shared common side effects, the reason for discontinuation was always compound specific and the toxicological or adverse effects which have motivated the discontinuation could be either due to the activation of PPARgamma, PPARalpha or both (class effect) or due to a PPAR unrelated effect. Thus, the risk evaluation of each adverse effect should be viewed on a case by case basis considering both the PPAR profile of the drug, its absorption/distribution profile, the nature of the side effect and the putative PPAR-related mechanism of action. This review mainly focuses on the preclinical and clinical adverse events of PPAR agonists that could be of concern when considering the development of new PPAR agonists. The selective modulation of PPAR activities is a promising approach to develop new drugs with preserved efficacy but diminished adverse effects.     

Citrus polymethoxylated flavones improve lipid and glucose homeostasis and modulate adipocytokines in fructose-induced insulin resistant hamsters

The present study was undertaken to determine whether supplementation with polymethoxylated flavones (PMFs) could ameliorate the fructose-induced hypertriglyceridemia and other metabolic abnormalities associated with insulin resistance (IR) in hamsters. Following feeding with the fructose diet, hamsters were supplemented orally with PMF-L or PMF-H (62.5 and 125 mg/kg/day) for 4 weeks. Both PMF-treated groups showed a statistically significant (p<0.05) decrease in serum triglyceride (TG) and cholesterol levels compared to the fructose-fed control group. The fructose control group at the end of the study showed elevated serum insulin and impaired insulin sensitivity (glucose intolerance). On the other hand, PMF-supplemented groups showed a reversal in these metabolic defects, including a decrease in insulin level and an improvement in glucose tolerance. PMF supplementation also reduced TG contents in the liver and heart and was able to regulate adipocytokines by significantly suppressing TNF-alpha, INF-gamma, IL-1beta and IL-6 expression and increasing adiponectin in IR hamsters. The mechanism of PMF on the activation of peroxisome proliferator-activated receptors (PPAR) was also explored. PMF-H supplementation significantly increased PPARalpha and PPARgamma protein expression in the liver. This is the first report of positive effects of PMF on adipocytokine production and on PPAR expression in IR hamsters. This study suggests that PMF can ameliorate hypertriglyceridemia and its anti-diabetic effects may occur as a consequence of adipocytokine regulation and PPARalpha and PPARgamma activation.     

Rosiglitazone prevents the impairment of human islet function induced by fatty acids: evidence for a role of PPARgamma2 in the modulation of insulin secretion

Peroxisome proliferator-activated receptors (PPARs) are a subgroup of the superfamily of nuclear receptors, with three distinct main types: alpha, beta and gamma (subdivided into gamma(1) and gamma(2)). Recently, the presence of PPARgamma has been reported in human islets. Whether other PPAR types can be found in human islets, how islet PPARgamma mRNA expression is regulated by the metabolic milieu, their role in insulin secretion, and the effects of a PPARgamma agonist are not known. In this study, human pancreatic islets were prepared by collagenase digestion and density gradient purification from nonobese adult donors. The presence of PPAR mRNAs was assessed by RT-PCR, and the effect was evaluated of exposure for up to 24 h to either 22.2 mmol/l glucose and/or 0.25, 0.5, or 1.0 mmol/l long-chain fatty acid mixture (oleate to palmitate, 2:1). PPARbeta and, to a greater extent, total PPARgamma and PPARgamma(2) mRNAs were expressed in human islets, whereas PPARalpha mRNA was not detected. Compared with human adipose tissue, PPARgamma mRNA was expressed at lower levels in the islets, and PPARbeta at similar levels. The expression of PPARgamma(2) mRNA was not affected by exposure to 22.2 mmol/l glucose, whereas it decreased markedly and time-dependently after exposure to progressively higher free fatty acids (FFA). This latter effect was not affected by the concomitant presence of high glucose. Exposure to FFA caused inhibition of insulin mRNA expression, glucose-stimulated insulin release, and reduction of islet insulin content. The PPARgamma agonists rosiglitazone and 15-deoxy-Delta-(12,14)prostaglandin J(2) prevented the cytostatic effect of FFA as well as the FFA-induced changes of PPAR and insulin mRNA expression. In conclusion, this study shows that PPARgamma mRNA is expressed in human pancreatic islets, with predominance of PPARgamma(2); exposure to FFA downregulates PPARgamma(2) and insulin mRNA expression and inhibits glucose-stimulated insulin secretion; exposure to PPARgamma agonists can prevent these effects.